In general, chemical modification of a specific protein residue is carried out to introduce a functional group onto the surface of a protein. Such chemical modification can be readily carried out and many specific residues can be modified at once, which is advantageous. On the other hand, excellent results are unlikely to be obtained in terms of reproducibility of the control of modified sites and/or the number of modified sites, which is problematic. Along with the recent developments in genetic engineering, it has become possible to substitute amino acid residues in proteins. Thus, it has become possible to introduce a desired unnatural amino acid having an amino skeleton into a protein by modifying a protein synthesis system, resulting in the realization of synthesis of a protein carrying functional groups with good reproducibility.
During protein synthesis, an amino acid first binds to the 3′ end of tRNA and then is transferred to a ribosome, where protein synthesis takes place. In a ribosome, translation from codons to amino acids takes place. With the use of tRNA bound to an unnatural amino acid, an unnatural amino acid can be incorporated into a protein.
The following method can be used for introducing an unnatural amino acid into a protein: a method wherein a codon at a target site for introduction is first substituted with a stop codon UAG and then translation is carried out in the presence of tRNA that has CUA as an anticodon and is aminoacylated with an unnatural amino acid (see Non-Patent Documents 1 to 4). In such a method, examples of tRNA used include tRNA for yeast phenylalanine (see Non-Patent Documents 1 and 2), tRNA for E. coli asparagine, tRNA for tetrahymena glutamine (see Non-Patent Document 3), and tRNA for E. coli glycine (see Non-Patent Document 4).
However, since tRNA that has CUA as an anticodon and is aminoacylated with an unnatural amino acid competes with a termination factor upon translation of UAG, the efficiency of introduction of an unnatural amino acid is not high in such case.
Non-Patent Document 1: Science, 244, p. 182, 1989
Non-Patent Document 2: Nucleic Acids Res., 18, 83-88, 1989
Non-Patent Document 3: Chem. Biol., 3, 1033-1038, 1996
Non-Patent Document 4: J. Am. Chem. Soc., 111, p. 8013, 1989